Identification of markers for migrasome detection

a Representative TEM images of negatively stained samples of migrasomes and exosomes purified from NRK cells overexpressing TSPAN4-GFP. Scale bar, 500？nm. b Representative cryo-EM images of migrasomes and exosomes purified from NRK cells overexpressing TSPAN4-GFP. Scale bar, 200？nm. c Overlap of proteins from migrasomes identified in three independent quantitative mass spectrum analyses. The central overlapping area shows the 4737 proteins that were reproducibly detected in all three analyses. 577 of these proteins have score >2 and ratio >1.5. d Overlap of proteins from exosomes identified in three independent quantitative mass spectrum analyses. The central overlapping area shows the 5375 proteins that were reproducibly detected in all three analyses.796 of these proteins have score >2 and ratio >1.5. e Venn diagram showing the number of proteins enriched on migrasomes and exosomes with score >2 and ratio >1.5 in all three repeats. The total number of proteins is shown above the diagram, and the unique proteins and shared proteins are shown in the circles. f Samples from cell bodies and purified migrasomes and exosomes were analyzed by western blotting using antibodies against Alix, Tsg101, Calnexin, Sec61α, GM130 and Tim23, and identified migrasome-specific marker NDST1, PIGK, CPQ and EOGT. g NRK cells stably expressing TSPAN4-GFP were cultured on a fibronectin-coated surface. NDST1, PIGK, CPQ and EOGT were detected by immuno-fluorescence using the corresponding antibodies. Scale bar: 5？μm. h Serum samples from three different individuals (1？ml/Sample) were subjected to centrifugation at 20000？×？g. The pellet was analyzed by western blot using antibodies against indicated proteins. i Fraction 7 from Supplementary Fig. 4B and exosomes purified from serum samples were analyzed by antibodies against Alix, Tsg101, Integrin β1, NDST1, EOGT, PIGK and CPQ. j Representative TEM images of negatively stained samples and embedded ultra-thin sections of migrasomes purified from human serum and from NRK cells stably expressing TSPAN4-GFP. Scale bars are indicated in the figur